Porous bacterial cellulose in cartilage tissue engineering

Jessica Andersson

Populärvetenskaplig sammanfattning

Artikulärt brosk har till uppgift att smörja lederna i våra knän samt absorbera stötar, vilket gör det till en mycket utsatt vävnad. Vävnaden saknar dock blodkärl, vilket leder till att skador inte kan självläka. Sedan 20 år tillbaka testas autologous chondrocyte transplantation (ACT) världen över för att försöka läka skador. Denna metod bygger på transplantation av expanderade celler från ett friskt till ett skadat område. På grund av begränsade resultat förekommer idag mycket forskning där celler tagna från biopsier får expandera in vitro för att sedan implanteras till det skadade området i olika material, s.k. scaffolds. Något optimalt material har ännu inte hittats och därför utforskas bakteriell cellulosa (BC) som ett alternativ.

Cellulosa är den mest förkommande biopolymeren som finns på jorden. Den finns i cellväggarna i trä och andra växter men produceras även naturligt av olika arter av alger, svampar och bakterier. Vissa arter hos bakterier producerar cellulosa-fibrer då de får tillgång till socker som kolkälla. Dessa bildar ett nätverk som liknar spindelnät vilket uppvisat många lovande egenskaper inom regenerativ medicin. Materialet har visat sig vara biokompatibelt, dvs. stöts inte bort från kroppen, ha bra mekaniska egenskaper, samt kunna tillverkas i en rad olika former, vilket gör det användbart i en rad applikationer.

För att gynna cellers migrering, proliferering och differentiering i materialet, har partiklar introducerats i materialet vid tillverkningen. Då dessa i efterhand tas bort, skapas ett mikroporöst material i vilket humana broskceller, kondrocyter, odlats. Dessa har visats fästa till materialet och i viss utsträckning även fylla upp porerna och börjat tillverka ämnen som ger brosket dess unika egenskaper och funktioner.

Tillverkningen av porös BC är idag ännu inte optimerad. Genom ytterligare utveckling av materialet kan detta i framtiden kunna komma att användas för att regenerera skadat brosk.

Examensarbete 30 hp

Civilingenjörsprogrammet Molekylär Bioteknik

Uppsala Universitet februari 2009

Jessica Andersson Executive summary

Cartilage lesions are among the most abundant defects occurring in society. Due to the non-self-healing nature of the tissue, they are very difficult to treat. Today, autologous chondrocyte implantation (ACI) is tested in random studies worldwide as a method for treatment. Insufficient results have put great demand on developing novel methods, in which various types of materials are tested as scaffolds for cartilage cells, chondrocytes. Therefore, the highly shapeable and biocompatible bacterial cellulose (BC) was tested as a candidate.

Successful attempts to make this material highly porous were made at the Biosynthetic Blood Vessel (BBV) Laboratory in Gothenburg. This was achieved by incorporating porogens during the fabrication of the material. Chondrocytes from multiple patients were introduced into the porous material with promising results. The cells attached to the material and furthermore, proliferated within it. In addition, cells migrated into the pores at some depth of the material, facilitating cartilage formation throughout a large volume. With further improvements, the porous BC show great potential as a material for the homing of chondrocytes.

BC should be considered an unique and excellent candidate as a scaffold material due to the enormous need for cartilage regenerative methods. By increasing the attachment capacity and the infiltration of cells into the whole volume of the material, this material will strongly compete with other materials on the market today. The possibility to fabricate the material into specialized shapes as well as the low cost at which the material is produced, are features that many of the materials on the market today, such as Hyaff

, lacks. Therefore, BC scaffolds should be further developed, making it possible to produce an off-the-shelf product which is easily manufactured and affordable worldwide. This could be done rather rapidly and inexpensive due to the ease of manipulating and changing the cultivation of the material.

Degree Project 30 hp

Master of Science in Molecular Biotechnology

Uppsala University February 2009

iv

1. INTRODUCTION ... 1

1.1 Bacterial Cellulose ...1

1.1.1 Structure and Assembly ... 2

1.2 Cartilage ...5

1.2.1 Composition, Structure and Properties ... 5

1.2.2 Cartilage Defects ... 10

1.2.3 Differentiation and Maintenance ... 11

1.3 Bacterial Cellulose in Cartilage Tissue Engineering ... 13

1.3.1 Scaffolds ... 13

1.3.2 Cell Seeding Techniques ... 15

1.4 Aim of Project ... 16

1.5 Analysis Methods ... 17

1.5.1 Fourier Transform Infrared Spectroscopy ... 17

1.5.2 Scanning Electron Microscope ... 18

1.5.3 Confocal Microscopy ... 19

1.5.4 Biochemical Analysis ... 20

1.5.5 Histology ... 20

2. MATERIALS AND METHODS ...24

2.1 Preparation of Paraffin Wax Spheres ... 24

2.2 Porous BC Networks ... 24

2.2.1 Packing of Particles ... 24

2.2.2 Pre-culture Acetobacter xylinum ... 24

2.2.3 Cultivation of Porous BC Scaffolds ... 25

2.2.4 Harvesting and Purification of Porous BC Scaffolds ... 25

2.3 Characterization of Porous BC Networks ... 26

2.3.1 Fourier Transform Infrared Spectroscopy ... 26

2.3.2 Scanning Electron Microscopy ... 26

2.3.3 Confocal Microscopy ... 26

2.4 Cell Study I ... 27

2.4.1 Expansion of Cells ... 27

2.4.2 Seeding of Cells onto Porous BC Scaffolds ... 27

2.4.3 Histology ... 28

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2.5.1 Expansion of Cells ... 28

2.5.2 Seeding of Cells onto Porous BC Scaffolds ... 28

2.5.3 Confocal Microscopy ... 29

2.5.4 Histology ... 30

2.6 Cell study III ... 30

2.6.1 Expansion of Cells ... 30

2.6.2 Seeding of Cells onto Porous and Regular BC Scaffolds ... 30

2.6.3 Confocal Microscopy ... 31

2.6.4 Biochemical Analysis ... 31

2.6.5 Scanning Electron Microscopy ... 32

3. RESULTS ...33

3.1 Production of Porous BC Networks ... 33

3.2 Characterization of Porous BC Networks ... 33

3.2.1 Fourier Transform Infrared Spectroscopy ... 33

3.2.2 Scanning Electron Microscopy ... 34

3.2.3 Confocal Microscopy ... 39

3.3 Criteria for Porous BC Scaffolds ... 40

3.4 Cell Study I ... 40

3.4.1 Histology ... 40

3.5 Cell Study II ... 42

3.5.1 Confocal Microscopy ... 42

3.5.2 Histology ... 43

3.6 Cell Study III ... 45

3.6.1 Confocal Microscopy ... 45

3.6.2 Scanning Electron Microscopy ... 51

3.6.3 Biochemical Analysis ... 55

4. DISCUSSION ...57

5. CONCLUSIONS...64

6. ACKNOWLEDGEMENTS ...65

7. BIBLIOGRAPHY ...66

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DMEM Dulbecco´s Modified Eagle´s Media EC Endothelial Cell

ECM Extracellular Matrix

FTIR Fourier Transmission Infrared GAG Glycosaminoglycan

MPC Metal Carpal Phalangeal OA Osteoarthritis

PBS Phosphate Buffered Saline PGA Poly-glycolic Acid

PLA Poly-lactic Acid PVA Poly-vinyl Alcohol

SEM Scanning Electron Microscopy SMC Smooth Muscle Cell

TC Terminal Complex

TEM Transmission Electron Microscopy

1

1. Introduction

There has been a lot of research within the field of cartilage tissue engineering throughout the last decades. Development of optimal scaffolds using various materials has aimed at finding an optimal material which allows cartilage cells, chondrocytes, to attach to and proliferate within the material. The work within this report has focused on using a highly porous scaffold made from bacterial cellulose to study the ability of chondrocytes to develop cartilage within the material.

1.1 Bacterial Cellulose

Cellulose is the most abundant natural occurring polymer on earth. It can be found in cell walls of plants and is synthesized naturally by some microorganisms of bacteria, algae and fungi. Cellulose has also been successfully produced in vitro by enzymatic polymerization as well as by chemical synthesis (chemosynthesis).

A summary of the various cellulose pathways can be found in figure 1.1.

Figure 1.1 Cellulose synthesizing pathways. Natural biosynthesis of cellulose is performed by plants and microorganisms, but can also be synthesized in vitro by enzymes and chemicals (adapted from Klemm et al.^{4, 5}).

Native celluloses are composites of two or more crystalline forms, especially cellulose I (α and β) and cellulose II.

Cellulose I contains cellulose chains which are arranged in parallel, whereas the chains in cellulose II have an antiparallel orientation.

PLANTS MICROORGANISMS

ENZYMATIC SYNTHESIS CHEMOSYNTHESIS

Ring-opening polymerization Biosynthesis and

removal of ligning and hemicelluloses

Polymer formation with purified cellulases

Biosynthesis by bacteria, algae and fungi

2

Kobayashi et al.

were the first to succeed in producing cellulose in vitro. They used an extracellular hydrolysis enzyme of cellulose (produced by Trichoderma viride) to artificially polymerize monomers of β-

-cellobiosyl fluoride into a cellulose derivative. However, this cellulose lacked the potential to “enable special functional groups to be introduced regiospecifically at the desired hydroxyl groups in the repeating pyranose units of cellulose”, and therefore, Nakatsubo et al.

developed a method to chemically synthesize cellulose having this advantage. They used 3,6-di-O-benzyl-α-

-glucopyranose 1,2,4-orthopivalate as monomers which by cationic ring-opening polymerization could be transferred into cellulose.

Cellulose has been known to be synthesized by bacteria since the 19

century, when Brown

discovered that the “Vinegar plant” was actually two components, bacteria and secreted cellulose. Especially the bacteria Acetobacter xylinum, synthesize and secrete extracellular cellulose with high versatility, high crystallinity, high water absorption capacity and good mechanical properties such as toughness, resilience and flexibility among other.

This makes the material attractive in many biomedical applications such as skin replacement, wound dressings, blood vessel substitutes and bone graft materials.

This type of cellulose, called bacterial cellulose (BC), has been used in recent studies to develop artificial blood vessels, cartilage and meniscus substitutes among other, showing biocompatibility and cell adhesion.

There are other types of bacteria that also produce cellulose, such as Rhizobium, Agrobacterium and Sarcina, although most studies have been performed using bacteria from the genus Acetobacter, especially the species xylinum.

1.1.1 Structure and Assembly

Bacterial cellulose is molecularly identical to naturally occurring plant cellulose. It is built up

by repeated units of

-glucose (C

H

O

) which are joined together by β(1→4)-

linkages, as

seen in figure 1.2.

Hydrogen bonding between chains of repeated

-glucose units form large

networks building up the cellulose structure. One major difference when comparing bacterial

cellulose to plant cellulose is that the latter is associated with hemicelluloses and lignin that

the former lacks.

This makes the bacterial cellulose a very pure natural polymer.

3

Figure 1.2 Chemical structure of bacterial cellulose subunits. Cellulose is built up by repeating units of

D-glucose (C6H12O6) which are liked together through β(1→4)-D linkages.

Acetobacter xylinum are gram-negative, obligate aerobic, rod-shaped bacteria about two μm long, which secrete cellulose extracellularly.

They form pellicles at the air-liquid surface which is thought to be done to help keeping the bacteria in an aerobic environment, to protect them from ultraviolet light and to prevent them from drying.

The assembly of these cellulose pellicles includes enzymatic steps in which glucose is transformed to UDP-glucose leading to formation of cellulose.

For this process, cellulose synthases are the essential enzymes.

These are located in the bacterial envelope, between the outer and the cytoplasma membrane, in so called synthesizing complexes or terminal complexes (TC).

Following is a brief description of the assembly of BC, which is also depicted in figure 1.3.

Firstly, sub-elementary fibrils with a width of about 1.5 nm are formed from glucan residues which are aggregated and elongated from the TC.

Through assembly of these sub- elementary fibrils, microfibrils are formed which are secreted through the surface of the bacterium.

About 50 of these microfibrils integrates into a ribbon of about 40-60 nm in width close to the surface of the bacteruim, and due to movement of the bacterium, the ribbon elongates with a rate of about 2 μm/minute.

Figure 1.3 Excretion of bacterial cellulose microfibrils and assembly into ribbons. Sub-elementray fibrils are formed within the bacterial envelope and assemblies into microfibrils which are secreted from the bacteria.

Outside, large ribbons are built up by the integration of a large number of microfibrils (adapted from Hirai et al.²²).

Sub-elementary fibril Microfibril

Ribbon assembly Acetobacter xylinum

4

Right before cell division of a parent cell, sub-elementary cellulose fibrils are started to be synthesized in both daughter cells.

When division has occured, and if the fibrils are kept intact, this continuous process of excretion of microfibrils give rise to the cross-linked pellicle network mentioned above with bacterial cells being embedded in between the ribbons. This process can also be seen in figure 1.4.

Figure 1.4 Pellicle formation in bacterial cellulose. One parent cell that is about to divide, starts to synthesize sub-elementary cellulose fibrils within both daughter cells before division. If these are kept intact throughout the whole cell division, a branched network, the pellicle, is being developed (adapted from Yamanaka et al.²³).

The production of bacterial cellulose in the laboratory is generally performed using Acetobacter xylinum grown on varying saccharides in a temperature of about 28-30 °C.

Furthermore, the cultivation media is often complemented with vitamines and trace metals to further improve the cultivation.

Cell

Cell division

Branching

Network formation

5 1.2 Cartilage

The human body contains three different types of cartilage, namely fibrous cartilage, elastic cartilage and hyaline cartilage.

Fibrous cartilage is a tough material which provides strength and support and can be found within the intervertebral discs, in the meniscus and at the end of tendons and ligaments, for instance.

Elastic cartilage, found in ears and noses, contains elastin which makes it stiff, yet elastic. Hyaline cartilage, which is usually referred to as articular cartilage, covers the ends of bones in diarthrodial joints, providing a near-frictionless motion and loading distribution.

Articular cartilage, further on referred to as cartilage, is a tissue with low healing capacity due to the tissue being avascular, aneural and alymphatic. This tissue is exposed to a lot of stresses throughout a life-time, due to the tissue functioning as a shock absorber within the joint.

Moreover, traumatic injuries and regenerative joint diseases are common within the tissue.

Due to the tissue having very low healing capacity, cartilage defects makes patients suffer and the attempts to heal the tissue becomes very expensive for society.

1.2.1 Composition, Structure and Properties

Articular cartilage, which covers the wear surfaces of diarthrodial joints and provides lubrication, is a biphasic connective tissue, with one fluid phase compost of water and electrolytes and one solid phase compost of chondrocytes, collagens, proteoglycans and other glycoproteins.

The ratio of these components within the tissue can be found in table 1.1. By allowing water to flow through the porous solid phase, cartilage acquires its mechanical and biochemical properties, such as high tensile strength (collagens) and resistance to compression (proteoglycans).

Table 1.1 Content of articular cartilage. Numbers refer to the amount of respectively component in wet state.²⁸

Water [%] Collagens [%] Proteoglycans [%] Chondrocytes [%]

≈ 60-85 15-22 4-7 ≈ 1

6

Cartilage is a highly structural tissue included within the macro-scale (0.5-15 cm) diarthrodial joint.

The cartilage itself is in tissue-scale (10

-10

m), compost of cells and extracellular matrix (ECM) in micro-scale (10

-10

m), collagens and proteoglycans in ultra-scale (10

-10

m) and charged groups in nano-scale (10

-10

m). This hierarchy provides cartilage with its unique and extraordinary properties and functions.

Collagens

More than 90 % of the collagens in cartilage are collagen type II.

Other types of collagens that are present in cartilage, but in minor amounts, are collagen type VI, IX and XI. All collagens are mainly built up by three polypeptide chains (α chains) of glycine, proline and X (where X is any amino acid or hydroxyproline), forming a right-handed triple helix, giving them their characteristic structure. These triple helices are further aggregating into larger collagen fibrils.

Some variations between the different collagens give them their unique function such as flexibility, stability and toughness.

Individual collagen fibers have diameters in a range of 20 to 200 nm and a length of 10 nm to 1 µm.

Through intramolecular and intermolecular cross-linking, collagens form a cohesive network providing cartilage high tensile stiffness and strength.

Proteoglycans

Proteoglycans are composed of long carbohydrate chains, glycosaminoglycans (GAGs) that

are being attached to a central protein core chain (figure 1.5).

These GAGs, the most

abundant ones being chondroitin sulphate and keratin sulphate, are negatively charged and are

therefore giving rise to a strong repulsive force between them, which causes the cartilage to

swell by keeping the proteoglycan molecules in a distended state.

This important feature

provides cartilage with compressive stiffness since the proteoglycans occupy such a large

volume that the tissue has lots of space which can be compressed.

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Figure 1.5 Schematic drawing of a proteoglycan monomer. Glycosaminoglycans, mainly chondroitin sulfate and keratan sulfate, are attached to a central core protein, building up a single proteoglycan. By aggregation of monomers to hyaluronan, larger proteoglycans are being formed.

There are many different proteoglycans in cartilage such as biglycan, decorin, fibromodulin, lumican and aggrecan, the latter being the most abundant one.

Proteoglycan monomers vary in length between 10 nm to 1 µm.

By forming aggregates with the carbohydrate hyaluronan as well as with collagen fibers, they help stabilizing the ECM of cartilage and provide the tissue with additional strength (figure 1.6).

Figure 1.6 Interactions between collagens and proteoglycans. The ECM of cartilage is being stabilized through interactions between collagens and proteoglycans, giving strength to the tissue (adapted from Mow et al.²⁸).

Hyaluronan Core protein

Link protein Hyaluronan

binding region Keratan sulfate rich region Chondroitin sulfate rich region

Chondroitin sulfate

Keratan sulfate

Monomer

Interstial fluid

Collagen fibril

Attached monomer Hyaluronan

40 nm

8 Chondrocytes

The cells of cartilage, chondrocytes, are located throughout the cartilage tissue in small compartments called lacunae. Only about 1 % of the tissue volume comprises chondrocytes.

They are about 15 µm in diameter and are responsible for cartilage formation, i.e. synthesis of collagens and proteoglycans and maintenance of the ECM.

This maintenance is being performed by cells responding to chemical and environmental factors that influence the ECM through which they also receive nutrients by diffusion.

Morphology and Structure

Cartilage composes four zones; the superficial zone, the middle zone, the deep zone and the calcified zone.

These are oriented as seen in figure 1.7, with the calcified zone being closest to the bone followed by the deep and middle zone and finally the superficial zone facing towards the joint. The building blocks of cartilage; water, collagens, proteoglycans and chondrocytes have varying features within these zones, leading to the unique properties of cartilage.

The superficial zone contains the largest amount of collagens as well as the smallest amount of proteoglycans as compared to the other zones.

Moreover, the superficial zone contains large amounts of water. The orientation of the collagens within this zone is parallel to the surface. This orientation is suggesting helping cartilage resist shear forces and compression as well as giving it high tensile strength. Chondrocytes within this zone have an elongated shape and are oriented along with the surface lining. These produce a superficial zone protein, lubricin, which provides lubrication to the diarthrodial joints.

Within the middle zone collagens are orientated in a more random fashion and fibers are

thicker than in the superficial zone.

Of all zones, this is the thickest zone with the

largest amount of proteoglycans. The density of cells is lower and the cells have a rounded

morphology. The random orientation of collagens and the high amount of proteoglycans,

together with the water content allows cartilage to sustain high loads and function as a shock

absorber.

9

Figure 1.7 Zone formation in articular cartilage. The calcified zone is being closest to the bone, followed by the deep zone, the middle zone and finally the superficial zone closest to the joint (adapted from Orthopaedic Research Center, Colorado State University³⁷).

The orientation of collagens in the deep zone is perpendicular to the underlying bone, with fibrils being inserted into the calcified zone and thereby anchoring the cartilage tissue into the underlying bone.

This zone contains a large amount of proteoglycans. Chondrocytes within this zone are oriented in a columnar fashion.

In the calcified zone the ECM is totally calcified with small chondrocytes being completely embedded within it, resulting in a very low metabolic activity.

The function of this zone is to transfer mechanical stresses from cartilage to bone.

The environment immediately surrounding the cells within cartilage also varies.

Closest to the cells is the pericellular matrix which has a large amount of proteoglycans and a small amount of collagens. Outside the pericellular matrix is the territorial matrix where thin

Chondrocytes

Longitudinally collagen fibers

Collagen fibers in cross section Superficial zone

Middle zone

Deep zone

Calcified zone

10

collagen fibers form a network protecting the cells. Together with the pericellular matrix, these matrixes bind the cell membranes, transmit mechanical signals to the chondrocytes and protect the cells when the cartilage deforms and is applied to loads.

The outermost matrix, the interterritorial matrix, contains large collagen fibrils which are oriented according to which structural zone the chondrocytes is present within, providing the tissue its mechanical properties.

1.2.2 Cartilage Defects

Mechanical, chemical and microbiological agents, such as traumatic injuries and regenerative joint diseases due to age and obesity for instance can damage cartilage, with one of the most common diseases being osteoarthritis (OA).

Taken to account that the tissue has a very limited healing capacity, this leads to difficulties when treating damages of the tissue. This makes cartilage defects very expensive for society and struggling for the patient.

In adult cartilage, the activity of chondrocytes and their ability to divide decline.

They continue to synthesize and counterchange the components of ECM, but with age, this turnover capacity decreases which can contribute to degeneration of the tissue. One treatment method which is tested today for cartilage lesions is autologous chondrocyte implantation (ACI), which is a cell based method.

This technique has been tested occasionally in some places since the 1980´s and has been performed on more than thousand patients in Sweden and more than 20 000 patients worldwide.

Yet, this is not a standardized method since only randomized clinical trials have been performed, making the outcome difficult to compare. In the first generation of this technique, called autologous chondrocyte transplantation (ACT), small cartilage biopsies from healthy, non load-bearing areas of the joints of patients were harvested and chondrocytes isolated using enzymatic digestion.

These were then cultured and expanded in vitro before being implanted to the damaged area where they were to produce new cartilage tissue, and sealed with a periosteal membrane. Figure 1.8 gives an overview of the technique used in ACT. This technique has had some problems with leakage and uneven distribution of cells within the defected area, leading to the development of improved techniques.

In the second generation ACI, the periosteal membrane has been replaced by cell-seeded

membranes, and in the third and developing generation, chondrocytes are being cultured in

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3D scaffolds.

The latter technique is similar to the first generation ACI, but takes longer time since isolated surplus chondrocytes are being cultured in scaffolds instead of in culturing flasks. This technique is being used by Brittberg and his colleagues, in Hyalograft

C scaffolds which are made from Hyaff

, a derivative of hyaluronic acid.

These scaffolds have shown good clinical results with hyaline-like repair tissue as a result.

Yet, no ideal scaffold has been found, leading to lots of research being focused on finding optimal cartilage mimicking scaffolds for achieving satisfactory cell proliferation and differentiation to use in tissue engineered cartilage for regenerative purposes.

An optimal future treatment method for cartilage defects would include the use of a off-the-shelf, arthroscopic biocompatible implant material with cells from a universal donor.

1.2.3 Differentiation and Maintenance

Differentiation is the process where cells undergo phenotypic changes to become a more specialized cell type.

Briefly, this process involves (i) lineage commitment and (ii) coordinated gene expression events, which ends up in the cells being differentiated into a new cell type. This process can sometimes be reversible, and is then referred to as de- differentiation. When de-differentiated cells are starting to differentiate again, the process is referred to as re-differentiation.

Cells respond differently to geometrically different biomaterial and behave differently on 2D and 3D substrates. For instance, chondrocytes de-differentiate when cultured on a 2D surface, whereas their phenotype is maintained when cultured in a 3D environment.

Recent studies have found that cells on substrates with controlled topography exhibit different behavior, suggesting cells could distinguish the geometry of the substrates such as shape or extent of roughness.

When differentiating, chondrocytes change their gene expression, an area in which lots of research is being performed.

The knowledge about genes that are being up- respectively down-regulated throughout this process, could be interesting when developing new therapeutic methods for cartilage defects.

By inducing and/or silencing the expression of specific genes using mechanical stimuli, specific molecules among others, the re-differentiation of chondrocytes within scaffolds could possibly be stimulated.

Alternatively, novel techniques for inducing cartilage regeneration in vivo could be

developed.

12

Figure 1.8 Autologous chondrocyte transplantation. Briefly, chondrocytes are harvested through arthrotomy and isolated using enzymatic digestion. The cartilage defect is prepared and a periosteal membrane is harvested.

After cultivation, chondrocytes are injected underneath the periosteal membrane and the would is closed using fibrin glue and stitches (adapted from Brittberg et al.³⁹).

Implantation of chondrocytes and wound closure Arthrotomy and

preparation of defect

Securing seal using fibrin glue and stitches Harvesting of periosteal flap

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1.3 Bacterial Cellulose in Cartilage Tissue Engineering

In the 1990s, Langer and Vacanti promoted the concept of tissue engineering, as a field in which the principles of biology and engineering were taken together to develop functional substitutes for damages tissue.

The failure of organs and tissues had become a “frequent, devastating and costly” problem in human health care, and with the use of tissue engineering, tissue creation and repair could possibly be solved.

1.3.1 Scaffolds

In cell-based tissue engineering, biomaterial scaffolds are used to accommodate cells at the implantation site to replace the lost or damaged ECM. A tissue-engineered scaffold is preferably fabricated as a 3D structure that allows cell attachment, migration and proliferation and thereby allowing them to carry out the functions as those of the native ECM.

Moreover, the scaffold should be (i) biocompatible, (ii) biodegradable, (iii) highly porous and have a large surface to volume ratio, (iv) mechanically strong, (v) shapeable and (vi) uniformly distributed with an interconnected pore structure.

Depending on the application, the criteria mentioned above are fulfilled in various ways, carrying out the specific needs. When designing a scaffold, one ought to keep in mind the structure of the tissue to be restored and the role of the ECM that eventually will replace the scaffold.

Cells that are used in cell-based tissue engineering come from a variety of sources.

They can be autologous, i.e. taken as a biopsy from the patient’s own tissue, allogenic, i.e. taken from a donor from the same species, syngenic, i.e. taken from a genetically identical donor, or xenogenic, in which cells are taken from a cross-species, i.e. animals cells being used in

humans.

Various materials to be used in cartilage reconstruction have been extensively studied over the

years, but none has yet been found to be ideal. Among the natural polymers studied are

alginate,

chitosan,

collagen,

hyaluronic acid,

and cellulose.

Synthetic polymers that

have been studied include poly-glycolic acid (PGA),

poly-lactic acid (PLA)

and poly-

vinyl alcohol (PVA).

14

Various techniques have been used when fabricating scaffolds, for instance particulate leaching, solvent casting, phase separation techniques, freeze drying, solid-free forming, 3D printing and combinations of these.

Furthermore, porogens of different materials such as paraffin, ice, gelatin, sugars, salt and agarose have been used.

A lot of research has been performed where BC has been used for scaffold fabrication and tissue engineering.

Endothelial cells (EC), smooth muscle cells (SMC) and chondrocytes have all shown to adhere to the material and in vivo studies have revealed that the material is non-toxic.

Svensson and co workers used unmodified respectively chemically sulfated and phosphorylated BC as scaffolds for bovine and human articular chondrocytes.

Results with transmission electron microscopy (TEM) showed that the unmodified BC supported chondrocyte proliferation and that cells migrated into the material to some extent, although the depth was only a few micrometers. The migration of cells deeper into the BC scaffolds was highly limited when Bäckdahl and coworkers

performed similar experiments. Therefore, a novel fabrication method was developed where fused paraffin porogens were incorporated into the cultivation of BC scaffolds.

By removing these afterwards, a highly porous and interconnected structure was obtained. SMC have been able to penetrate this porous material in vivo to a somewhat larger extent than in previous experiments, showing potential in tissue engineering.

The novel technique of introducing porosity into BC scaffolds mentioned above includes paraffin porogens which are incorporated into the cultivation process of Acetobacter xylinum.

These porogens are placed around a silicone tube which is placed in the middle of a bioreactor and allowed to melt together to some extent by placing the bioreactors into a warm water bath. After addition of cultivation media and inoculation of the bacteria, the bioreactors are connected to an oxygen flow, creating an air-liquid surface around the permeable silicone tube. This will cause the bacteria to start grow at this surface, creating a BC material with the shape of a tube. A schematic drawing of the setup used can be seen in figure 1.9.

Studies on this material have revealed that the inner sides of the tubes are much denser than

the outer sides, possible being due to the fact that more bacteria are being active immediately

at the air-liquid surface as compared to at some distance from it.

For usage as scaffolds,

these BC tubes are therefore being cut open and material of desired shape and size cut out.

15

Figure 1.9 Schematic drawing of bioreactor setup. Paraffin wax spheres are placed around a permeable silicone tubing and allowed to melt together to some extent. Bacteria are added and these grow around the permeable silicon tubing when the bioreactor is connected to an oxygen flow, forming a BC tube with paraffin wax spheres introduced into the fiber network (adapted from Bäckdahl et al.⁶⁵).

1.3.2 Cell Seeding Techniques

One of the most important aspects to consider when it comes to engineer various tissues is the cell seeding technique used. A successful technique involves (i) a high efficiency, i.e. the ratio of attached cells to seeded cells, (ii) uniform distribution of cells throughout the scaffold and (iii) a high cell survival number.

These factors influence the proliferation of cells, the formation of extracellular matrix as well as the overall functionality of engineered tissue.

Attachment of cells to the ECM is performed by transmembrane proteins named integrins.

These recognize specific peptides which they bind to. These peptides, for instance the RGD peptide, could therefore be used on scaffolds to increase the adhesion capacity of cells to the scaffold surface.

Furthermore, cells are known to favor hydrophilic surfaces over hydrophobic.

Lots of seeding techniques are presented in the literature and include static as well as dynamic approaches. Important issues to consider for a desirable seeding of cells are the cell seeding density, the hydrodynamic and/or mechanical environments and the physical and chemical

Oxygen

Silicone plug Glass tube

Silicone tubing

Paraffin wax spheres

Metal anchor

16

properties of the scaffold among other.

Static seeding techniques that have been used include applying of capillary action, centrifugal force and pressure, as well as varying cell seeding density and scaffolds properties such as hydrophilicity and porosity.

. Among the dynamic seeding techniques being used are spinner flasks, vacuum chambers, perfusion chambers and agitation.

The results of all these studies have been varying, proving the difficulties of biological systems. A method that is better in a certain application may not necessarily be the most advantageous to use in another one. Overall, the importance is to find a technique which allows a certain cell type to become evenly distributed within a certain scaffold and supply them with appropriate nutrients to maintain their function.

As mentioned above, the material used for the studies in this thesis has two different morphologies when comparing the inner and outer side of the BC tubes forming the scaffolds.

It seems logical that the outer, more porous side would be the most efficient side to seed chondrocytes on, since previous studies on regular, non-porous BC has shown limited cell ingrowth

. Moreover, the usage of an undertow could help forcing cells into the pores of this highly porous scaffold material. Therefore, different seeding techniques will be used in this thesis to try to reveal the behavior of chondrocytes within the porous BC scaffolds.

1.4 Aim of Project

The aim of this project was to optimize production of, and characterize, porous BC scaffolds and investigate its potentials as scaffold material in cartilage tissue engineering. With the material being highly porous, the ambition was that adult human articular chondrocytes would be able to attach to the porous BC and proliferate and redifferentiate within the interconnected scaffold material.

Porous BC scaffolds with controlled pore sizes were fabricated and characterized using FTIR,

SEM and confocal microscopy. This was followed by cell culture experiments to (i) study the

homing of chondrocytes within the material and (ii) to optimize the seeding of cells onto it.

17 1.5 Analysis Methods

In the following section, the principles of the analysis methods used within this thesis are described. The section ends with an experimental flow chart of this project.

1.5.1 Fourier Transform Infrared Spectroscopy

The molecular content in samples can be studied using Fourier transform infrared (FTIR) spectroscopy.

This method uses mid-infrared radiation (wavenumber between about 4 000 and 400 cm

) which is radiated through a sample, allowing the energy to be absorbed within it, causing the chemical bonds within the material to vibrate. Functional groups within the sample have a tendency to absorb in the same wavenumber length, regardless the structure of the whole molecule. Due to this feature, it is possible to detect functional groups within a sample and comparing the spectra obtained with spectra of known materials. This way, the chemical components within an unknown sample can be determined.

Figure 1.10 FTIR spectra for paraffin respectively BC controls.

The FTIR spectra for BC as well as for paraffin can be seen in figure 1.10. Natural cellulose has some characteristic peaks around 1 600, 2 900 and 3 400 cm

, whereas paraffin has characteristic peaks around 720, 1 450 and 2 900. By obtaining spectra of purified BC and comparing them with the spectra for pure BC and paraffin, conclusions can be drawn about the chemical composition of the sample.

4000 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400 3400

2900 1638

720 Paraffin

Wavenumber [cm^-1] 1450

BC

18 Advantages

One great profit of using FTIR spectroscopy is that it is a rapid method to achieve lots of information regarding the sample of interest.

Furthermore, the preparation of samples is relatively easy to perform and the analysis quite inexpensive.

Disadvantages

The major drawback of FTIR spectroscopy is that it does not give any information about the atoms, due to single atoms not containing any chemical bonds which can absorb radiation and thereby start to vibrate.

Of the same reason, it is impossible to detect noble gases and homonuclear diatomic molecules with FTIR spectroscopy. Furthermore, FTIR spectroscopy is limited when it comes to analyzing highly complex samples, since these will contain various functional groups, leading to difficulties when interpreting the spectra of the analyses.

1.5.2 Scanning Electron Microscope

Scanning electron microscopy (SEM) is an analysis method where the surface topography of samples are studied by scanning them with a beam of high-energy electrons, giving rise to a detailed 3D picture.

This can be performed on various heterogeneous materials in the range of nanometer to micrometer scale. The concept of the technique was first described by Max Knoll in 1937.

This technique uses electrons which are emitted from a cathode (firstly made from tungsten, but now other materials such as LaB

and gold are being used) of an electron gun, by applying a high voltage.

The electron beam is focused (about 10 nm spot size) using electron lenses and interacts with the sample, causing secondary electrons and backscattered electrons from the sample to be emitted and detected by specialized detectors. This is being performed vacuum chambers, since the mass of electrons is so small and otherwise would be scattered by air molecules. The amount of electrons being emitted from each scanned area, the intensity, determines the brightness of the picture, building up topographic image in grey-scale.

The samples studied have to be electrically conductive to be able to emit secondary

electrons.

Therefore, biological samples for instance, needs to be sputter coated with some

material that is conductive. Usually, this material is gold, palladium or platinum, but there are

other materials used. Briefly, the coating material is being exposed to energized gas plasma

19

formed from a heavy inert gas such as argon. This bombardment causes atoms to eject from the surface of the material, which collide with gas molecules, leading to a coating being formed on the specimen.

Furthermore, for the samples structure to be kept intact and to prevent degradation, biological samples are often fixed.

This is usually being done by placing samples in glutaraldehyde, such as in Karnovsky fixative solution.

Advantages

One great advantage with SEM is that is can be applied onto various materials, giving high resolution images of the most bulky materials.

Furthermore, the technique has a large depth of field, giving rise to the 3D appearance mentioned earlier.

Disadvantages

The drawbacks with this method include the preparations needed to make specimens electronically conducted as well as the preparations needed to enable analysis of biological samples. Another disadvantage is that only the surface of the specimen is studied.

1.5.3 Confocal Microscopy

Confocal microscopy is a microscopy technique that was commercialized in 1987, where samples are studied in extremely clean, thin optical sections.

This is achieved by a pinhole which removes out-of-plane light, giving rise to high resolution images of a sample. The method uses laser light of various wavelengths which are being point illuminated to the specimen. This causes electrons at this point in the sample to excite and fluoresce or the light to reflect (depending on the mode of action). By detecting the light that is not being obstructed by the pinhole, and by scanning the sample plane by plane, it is possible to reconstruct the 3D structure of the specimen studied.

Advantages

One of the advantages of using confocal microscopy is that it gives rise to high resolution 3D

images of tissues.

It is also possible to scan deeply into the sample of interest using different

objectives which enables larger 3D image constructs to be achieved as compared to for

instance SEM, where only the topography of a surface can be obtained.

20 Disadvantages

Since the technique measures very thin sections, the fundamental limits are related to the quantitative accuracy of the analysis being made.

The rate of scanning, the resolution of the image and the amount of light being exposed to the specimen determines the efficiency of the analysis.

One further disadvantage of this technique is that it is highly complex, making it somewhat difficult to use. Lots of knowledge needs to be known about the specimen, such as excitation and emission wavelengths, which can be difficult to determine for highly complex materials.

Furthermore, overview pictures and larger areas of uneven surfaces (such as BC) are hard to make focused.

1.5.4 Biochemical Analysis

To determine the amount of DNA found within a cell seeded scaffold construct, biochemical analysis is being performed. This method is mainly composed of two steps, (i) extraction of DNA and (ii) determination of the amount of DNA using a fluorescent dye. Papain, which is a protease breaking peptide bonds, is being added to the scaffold. This will break down the ECM within the scaffolds, extracting the DNA. By adding Hoechst stain, which is a fluorescent stain binding to DNA; spectroscopy can be used to determine the amount of fluorescent within each sample. This is being done by comparing the intensity of fluorescence with reference samples made. In the protocols used in the experiments performed throughout this thesis, reference samples are made from calf thymus.

1.5.5 Histology

The study of the microanatomy of cells, tissues and organs is referred to as histology.

To

perform a histology analysis, the sample of interest firstly needs to be fixed to preserve its

structure. This is often being performed chemically and the most commonly used fixative is a

formaldehyde solution, of which Histofix

is one of them, also being used within the

experiments being performed within this thesis. Formaldehyde reacts with the amino groups

of proteins forming methylene bridges between them, thereby preserving the structure of cells

and ECM components. Another way to fix a sample is what is referred to as cryo-

21

preservation, a method in which the sample is freeze in a special fixative to stabilize the structure of the tissue components.

The second step is to embed the sample to permit sectioning of it.

This is usually being done by dehydrating the sample using alcohol and thereafter embedding the sample in for instance paraffin or plastics. Thereafter, the sample is mounted and the sample is sliced in thin sections using a microtome. The obtained slices are thereafter placed on objective glasses, rehydrated and stained to enable the identification of various components within the tissue. When using the cryo-preservation technique, slicing is performed on the freeze material and slices are thereafter placed onto objective glasses, let to dry and stained. Both methods are thought to give identical results, with the method chosen being dependent on the material studied (e.g.

the scaffold material or tissue).

For this thesis, the collagen and proteoglycan content within our cell-seeded scaffolds is studied. This is being performed by staining the sample slices with Alcian blue-van Gieson, which stains GAG´s and collagens, respectively.

Previous experiments

Previous attempts to make histology analyzes of chondrocyte seeded porous BC scaffolds

showed that the preservation technique used had great impact on both the staining of the

prepared slices and the extent to which the scaffolds held together when sliced (Hanna

Stenhamre, unpublished results). Both methods gave rise to slices which easily fell apart

(figure 1.11 and 1.12) leading to difficulties when drawing conclusions regarding the cells

incorporation in the material. The main problem with the BC material is that it is very soft,

comparable to gelatin, even after fixation and embedding. The slicing of the material therefore

becomes very difficult, leading to slices where the material often falls apart.

22

(a) (b)

Figure 1.11 Cryo-preserved chondrocyte seeded porous BC scaffolds (Hanna Stenhamre, unpublished results).

The tissue is stained with Alcian blue-van Gieson. Pink/purple staining indicate collagens whereas blue staining indicate GAGs. Cell nuclea are seen as black spots. The scaffold material as well as the tissue components are very difficult to distinguish.

When staining slides from cryo-preserved scaffolds, the colors became too bright and crystal- like which made it hard to separate between cell nucleus and other components in cartilage.

Comparing this with the staining of paraffin-preserved scaffolds, the staining in the later technique became much better with cells, proteoglycans and collagens being distinctively stained. Furthermore, the scaffold material was much easier to distinguish within scaffolds that had been preserved using paraffin. Therefore, conclusions were drawn that scaffolds used in this thesis project were to be fixed in Histofix

and embedded in paraffin before stained for specific components.

Figure 1.12 Paraffin-preserved chondrocyte seeded porous BC scaffolds (Hanna Stenhamre, unpublished results). The tissue is stained with Alcian blue-van Gieson. Pink staining indicate collagens whereas blue staining indicate GAGs. Cell nuclea are seen as black spots. The light blue area in the figure is the cellulose scaffold. As seen in the figure, the material has fallen apart with chondrocytes being attached to the edges of the material.

23

Figure 1.13 Flow chart of BC scaffold production and cell cultivation. Scaffolds of porous BC are fabricated using paraffin porogens being incorporated into the cultivation of Acetobacter xylinum. After purification and removal of porogens, human articular chondrocytes are seeded onto the scaffolds and cultivated. Analysis of proliferation and re-differentiation determine the efficiency of the scaffold material.

Surfactant Ethanol

Proliferation and re-differentiation of chondrocytes in porous

BC scaffold Expanded

articular chondrocytes

from biopsy

Porous BC scaffolds Purification of porous

BC scaffolds Pre-culture of

Acetobacter xylinum

Cultivation media Cultivation of porous BC

in bioreactors Sieving and sterilization of formed paraffin wax spheres Melted paraffin sprayed into

PVA/water solution

Cultivation media

24

2. Materials and Methods

2.1 Preparation of Paraffin Wax Spheres

Paraffin wax (Joel Svenssons Vaxfabrik AB, Ljungby, Sweden) was placed into a water bath of 90 °C to allow it to melt. Polyvinyl alcohol (PVA) powder (5 g/l; Sigma-Aldrich

, Steinheim, Germany) was dissolved in stirring deionized water and the solution was heated to a temperature of 90 °C. The melted paraffin was poured into the heated PVA/water solution to form particles using a syringe, as described elsewhere.

The particle solution was then sieved and particles of sizes 90-150 µm, 150-300 µm and 300-500 µm were collected.

To sterilize the collected particles, these were immersed in an ethanol:water mixture (70:30).

The particles where then placed in a freezer for 24 hours before freeze drying (PowerDry PL3000, Heto).

2.2 Porous BC Networks

2.2.1 Packing of Particles

Sterilized particles were poured carefully into half of the volume of 70 ml glass tubes with one silicone tube with a diameter of 6 respectively 8 mm (AdvantaPure, NewAge Industries, Southampton, PA, USA) positioned in the middle (figure 1.9).

Thereafter the glass tubes were placed into a water bath of 40 °C for 40 minutes to allow the porogens to melt together.

When cooled to room temperature, tubes were ready for cultivation.

2.2.2 Pre-culture Acetobacter xylinum

100 ml of culturing media, adapted from Matsuoka et al.,

(fructose [40 g/l], yeast extract [5 g/l], (NH

)

SO

[3.3 g/l], KH

PO

[1 g/l], MgSO

·7H

O [0.25 g/l], corn steep liquor [20 ml/l], trace metal solution [10 ml/l, (EDTA [3 g/l], CaCl

·2H

O [1.47 g/l], FeSO

·7H

O [0.36 g/l], Na

MoO

·2H

O [0.242 g/l], ZnSO

·7H

O [0.173 g/l], MnSO

·5H

O [0.139 g/l]

and CuSO

·5H

O [0.005 g/l])] and vitamin solution [5 ml/l, (vitamin B

[40 g/l], vitamin B

[8 g/l], vitamin B

[8 g/l], vitamin B

[8 g/l], vitamin B

[4 g/l], vitamin B

[4 g/l], vitamin B

25

[4 g/l], vitamin B

[0.04 g/l] and vitamin B

[0.004 g/l])]) with a pH of 5.5 was poured into a Rough flask and autoclaved. 0.5 ml of vitamin solution was added since these were destroyed during the autoclave process. One aliquot of Acetobacter xylinum subsp. Sucrofermentas, BRP2001, trade number 700 178

, purchased from the American Type Culture Collection (LGC Promochem AB, Borås, Sweden) was thawed at room temperature and inoculated into the Rough flask (prepared as described by Bodin et al.

). The Rough flask was then placed horizontal in an incubator of 37 °C for 2 days.

2.2.3 Cultivation of Porous BC Scaffolds

Bacteria were loosened from the produced biofilm pellicle by shaking the Rough flask. 2.5 ml of bacteria suspension (cell density about 3.7x10

cfu/ml) was added to each glass tube and tubes were thereafter filled with culturing media (recipe described in section 2.2.2) repeatedly until all paraffin porogens were soaked. Tubes were then put into an incubator of 30 °C and connected to an oxygen flow of about 100 ml/min. Cultivation was performed during 7 days.

2.2.4 Harvesting and Purification of Porous BC Scaffolds

Fermented porous BC tubes were removed from the silicone tubing and excess particles removed by rinsing the tubes in deionized water. Tubes where then placed in deionized water in a water bath with a temperature of 90 °C for 1 hour to melt away some of the particles. To destroy and remove the bacteria stuck in the cellulose network; tubes were placed in 0.1 M NaOH over night. Thereafter the tubes were purified by placing them in fresh 0.1 M NaOH for 4 hours in a water bath of 60 °C followed by rinsing them twice in deionized water in the same temperature for 2 hours respectively.

Extensive purification of the porous BC tubes was performed by placing the tubes in 1 vol%

of the surfactant Berol EZ-1 (Akzo Nobel, Stenungsund, Sweden) over night in a shaking

water bath of 75 °C. Tubes where then rinsed in deionized water 3 times followed by

purification in ethanol for 8 hours in a shaking water bath of 75 °C. This procedure was

repeated until no porogens were visible under a light microscope and no residues could be

seen in a FTIR spectrum.

26

2.3 Characterization of Porous BC Networks

When fabricating porous BC, the obtained material appeared very different from time to time.

Some were thicker than other and the porosity within the scaffolds varied. Therefore, different techniques were used to study the scaffolds so that criteria could be set up for the porous BC scaffolds to use in cell studies.

2.3.1 Fourier Transform Infrared Spectroscopy

Samples from porous BC tubes were autoclaved and let to dry on glass slides inside a LAF bench. A small amount from each sample was scraped off and mixed with KBr and the transmission measured (PERKIN ELMER System 2000 FTIR). Comparison with spectra from samples of the paraffin used and BC (figure 1.10) revealed whether or not the porous BC tubes were sufficiently purified.

2.3.2 Scanning Electron Microscopy

Samples from porous BC made with the three different porogen sizes were placed in DI water in Petri dishes. These were then placed in liquid nitrogen to allow the samples to rapidly freeze. Thereafter the samples were placed in the freezer over night before freeze-dried. After being freeze-dried, samples were mounted and sputtered (EMITECH K550X). Samples were then analyzed using SEM (ZEISS CSM 982 GEMINI) to study the porosity and interconnectivity of the pores within the material.

2.3.3 Confocal Microscopy

When fabricating the porous BC material, the paraffin spheres were melted together as

described previously, to prepare an interconnected structure. To confirm this

interconnectivity, fluorescent microspheres (FS07F, diameter 19.11 µm, λ

: 441 nm

(yellow); λ

: 486 nm (green); Bangs Laboratories Inc., Fishers, IN, USA) with

a concentration of about 1x10

microspheres/ml were added onto the material and allowed to

migrate into the scaffold statically, mimicking cells migration through the scaffold.

Scaffolds were then studied using the confocal microscope (inverted ZEISS LSM 510

META), to determine the degree of interconnectivity within the scaffolds.

27 2.4 Cell Study I

An overview of the number of scaffolds and analysis methods used for the experiments described in this section is found at the end of section 2.6, table 2.1.

2.4.1 Expansion of Cells

Surplus chondrocytes from a patient (born 1986) undergoing ACI were expanded and cultured in culturing media (Dulbecco´s modified eagle´s media (DMEM)/F12 [Invitrogen, Grand Island, NY, USA] supplemented with L-ascorbic acid [0.025 mg/ml; Apotekets produktionsenhet Umeå, Sweden], gentamicin sulphate [50 mg/l; Gibco, Scotland], amphoterricin B [250 μg/ml; Gibco], L-glutamine [2 mM; Gibco] and 10 % human serum).

The chondrocytes were harvested with trypsin-EDTA solution (trypsin [0.125 %, Invitrogen]

diluted in 0.1 M phosphate buffered saline (PBS) [PAA Laboratories, Pasching, Austria] with EDTA [0.2 g/l]) when the cells had reached a confluence of at least 80 % (approximately 7 days).

Figure 2.1 Chondrocytes cultured in a 2D environment, about 100 % confluence. Chondrocytes receive an elongated morphology when cultured in 2D as compared to a more round morphology when cultured in 3D.

2.4.2 Seeding of Cells onto Porous BC Scaffolds

As a pilot study, chondrocytes were seeded onto the outer, more porous, side of porous

bacterial cellulose scaffolds. Four scaffolds with pore size 150-300 µm and a seeding surface

area of about 0.5 cm

were seeded with approximately 2x10

cells onto each square centimeter

of the scaffolds. The cell containing scaffold constructs were cultured in a pellet culturing

media (DMEM high glucose [PAA Laboratories, Linz, Austria] supplemented with ITS+

28

[Life Technologies, Sweden], linoleic acid [5.0 μg/ml; Sigma-Aldrich, Stockholm, Sweden], human serum albumin (HSA) [1.0 mg/ml; Equitech-Bio, TX], TGF-β1 [10 ng/ml; R&D Systems, U.K.], dexamethasone [10

M; Sigma], ascorbic acid [14 μg/ml; Sigma] and 1x penicillin-streptomycin [PAA Laboratories, Linz, Austria]). The media was exchanged every third day, and the cultivation performed throughout 21 days.

2.4.3 Histology

After 21 days of culturing, the four scaffolds were rinsed 2x5 minutes with Dulbecco´s PBS (PAA Laboratories, Pasching, Austria) and placed in Histofix

(Histolab Products AB, Gothenburg, Sweden) for 24 hours. Scaffolds were then placed in ethanol and sent to Histocenter (Gothenburg, Sweden) where they were dehydrated, embedded in paraffin, rehydrated and sliced (7 µm thickness) before stained with Alcian blue van Gieson to identify cells and proteoglycans respectively in the scaffolds. Histology slices where thereafter studied under the microscope (Nikon ECLIPSE 90i).

2.5 Cell Study II

An overview of the number of scaffolds and analysis methods used for the experiments described in this section is found at the end of section 2.6, table 2.1.

2.5.1 Expansion of Cells

Surplus chondrocytes from a patient (born 1992) undergoing ACI were expanded, cultured and harvested using the same protocol as described under Cell Study I.

2.5.2 Seeding of Cells onto Porous BC Scaffolds

Chondrocytes were seeded onto scaffolds with pore sizes of 150-300 µm using three different

techniques; (i) seeding through the cross section of the scaffold, (ii) onto the inner, less porous

side of the scaffold, and (iii) onto the outer, more porous side of the scaffold. The first seeding

technique was performed by placing scaffolds in between to object glasses which were

separated by aluminum foil and held together with metal clips. This gives rise to undertow,

which together with gravity helps pushing the cells into the material (see figure 2.2 for setup).